Versatile Biomaterial Additive: A Game-Changing Multifunctional Synthetic Peptide With Pro-Regenerative, Anti-Inflammatory, and Antibacterial Properties

The prevalence of bone disorders and deformities is increasing due to trauma, malignant neoplasms, infections, and degenerative and inflammatory diseases. Bone repair and replacement have evolved as a consequence of advancements in orthopedic technology and biomaterials with enhanced properties. The rapid growth of the bone tissue engineering field is being significantly influenced by biomaterials, such as polymer scaffolds with appropriate surface modifications. New additives are constantly being developed in response to the increasing demand for enhancing the bioactivity of biocomposites used for bone regeneration. We present the design and synthesis of a synthetic bioactive peptide UG46, which is multifunctional and consists of the fragment of human Cystatin C (CystC) and anoplin. In addition, the peptide is assessed as an additive that is employed to enhance the repair of bone by enriching porous chitosan (CH) scaffolds. Our results indicated that the UG46 peptide possesses pro-regenerative properties, while it did not exhibit any cytotoxic effects on human osteoblasts or human fibroblasts. Incubation of cells with CH and CH-UG46 extracts significantly increased cell proliferation, cell proliferation over 200% of control cells. Even though the proliferation assay revealed a significant inhibition of cell proliferation in cells seeded directly on the composites, the beneficial effect of the UG46 peptide was still noticeable. Additionally, the UG46 peptide exhibited dose-dependent anti-inflammatory properties in both its free form and as a biocomposite additive, qualifying it as a promising candidate for a bone biomaterial component. The working concentrations of UG46 peptide have been established at 40–80 μg/mL. The molecular structure analysis of the CH-UG46 biocomposites revealed that the majority of the pores were sufficiently large to enable osteoblast cells to infiltrate the scaffold, while concurrent microporosity (< 20 μm) enabled cell infiltration, vascularization, and cell-matrix interactions. Additionally, the peptide alone exhibits limited antibacterial properties; however, the peptide released from the CH-UG46 biocomposite at high levels has been demonstrated to be capable of inhibiting the growth of the selected bacterial strains that are most frequently found infecting healing wounds by over 83% for all strains tested stains with A. baumani bacterial count reduction at 99.86%. Our findings indicate that the bioactive peptide we have proposed is a promising enhancement for porous scaffolds. It has the potential to facilitate the creation of specialized, custom-designed biomaterials with multifunctional properties, including healing, defense, and hygiene.

Design and Characterization of Antibacterial Peptide Nanofibrils as Components of Composites for Biomaterial Applications

Purpose: The purpose of this study was to design and synthesize the ug46 peptide, incorporate its fibrils into composite materials, and evaluate its structural and antimicrobial properties. Another objective was to utilize spectroscopy and molecular simulation, enhanced by Machine Vision methods, to monitor the aggregation process of the ug46 peptide and assess its potential as a scaffold for an antimicrobial peptide.

 Method: The structural analysis of the ug46 peptide reveals its dynamic conformational changes. Initially, the peptide exhibits a disordered structure with minimal α-helix content, but as incubation progresses, it aggregates into fibrils rich in β-sheets. This transformation was validated by CD and ThT assays, which showed decreased molar ellipticity and an increase in ThT fluorescence.

Results: Laser-induced fluorescence and molecular dynamics simulations further revealed the transition from a compact native state to extended “worm-like” filament structures, influenced by peptide concentration and temperature. TEM and AFM confirmed these changes, showing the evolution of protofibrils into mature fibrils with characteristic twists. When incorporated into chitosan- bioglass composites, these fibrils significantly enhanced antimicrobial activity against pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa.

 Conclusion: Overall, ug46 peptide fibrils show promise as a multifunctional scaffold with structural and antimicrobial benefits in composite biomaterials.

Bioactive glasses enriched with zinc and strontium: synthesis, characterization, cytocompatibility with osteoblasts and antibacterial properties

Purpose: The aim of the presented work was to characterize the new obtained bioglasses and assess their biological performance in vitro. Bioglasses were produced using the sol-gel method in the SiO₂-P₂O₅-CaO system, for the purpose as composite ingredients. Their chemical composition was enriched with ZnO to introduce antibacterial properties and SrO with osteoinductive effect. The properties of bioglasses were compared and the effect of chemical composition and particle size on their biological properties was assessed. Methods: The bioglasses were evaluated via TG-DTA, FTIR, SEM-EDS analyses before and after incubation in SBF solution. LDH and WST-1 tests were used to determine the level of cytotoxicity of the tested bioglasses on hFOB1.19 osteoblasts. Results: The results show that the developed bioglasses release Ca²⁺, are bioactive in SBF solution, not cytotoxic and show antibacterial activity in contact with Pseudomonas aeruginosa and Staphylococcus aureus strains. Bioglasses enriched with ZnO show the highest bactericidal activity. All tested bioglasses enhanced hFOB 1.19 cells proliferation. Particle size has a lower effect on biological performance of the bioglasses than their chemical composition. Conclusions: The conducted research showed that bioglass modification with SrO and ZnO can be considered particularly for the development of biomaterials supporting bone regeneration and the treatment of infected bone defects

Effect of Selected Crosslinking and Stabilization Methods on the Properties of Porous Chitosan Composites Dedicated for Medical Applications

Chitosan is one of the most commonly employed natural polymers for biomedical applications. However, in order to obtain stable chitosan biomaterials with appropriate strength properties, it is necessary to subject it to crosslinking or stabilization. Composites based on chitosan and bioglass were prepared using the lyophilization method. In the experimental design, six different methods were used to obtain stable, porous chitosan/bioglass biocomposite materials. This study compared the crosslinking/stabilization of chitosan/bioglass composites with ethanol, thermal dehydration, sodium tripolyphosphate, vanillin, genipin, and sodium β-glycerophosphate. The physicochemical, mechanical, and biological properties of the obtained materials were compared. The results showed that all the selected crosslinking methods allow the production of stable, non-cytotoxic porous composites of chitosan/bioglass. The composite with genipin stood out with the best of the compared properties, taking into account biological and mechanical characteristics. The composite stabilized with ethanol is distinct in terms of its thermal properties and swelling stability, and it also promotes cell proliferation. Regarding the specific surface area, the highest value exposes the composite stabilized by the thermal dehydration method.

The Preliminary Assessment of New Biomaterials Necessitates a Comparison of Direct and Indirect Cytotoxicity Methodological Approaches

Background: Cytotoxicity testing is a primary method to establish the safety of biomaterials, e.g., biocomposites. Biomaterials involve a wide range of medical materials, which are usually solid materials and are used in bone regeneration, cardiology, or dermatology. Current advancements in science and technology provide several standard cytotoxicity testing methods that are sufficiently sensitive to detect various levels of cellular toxicity, i.e., from low to high. The aim was to compare the direct and indirect methodology described in the ISO guidelines UNE-EN ISO 10993-5:2009 Part 5. Methods: Cell proliferation was measured using WST-1 assay, and cytotoxicity was measured using LDH test kit. Results: The results indicate that the molecular surface of biomaterials have impact on the cytotoxicity and proliferation profile. Based on these results, we confirm that the indirect method does not provide a clear picture of the cell condition after the exposure to the surface, and moreover, cannot provide complete results about the effects of the material. Conclusions: Comparison of both methods shows that it is pivotal to investigate biomaterials at the very early stages using both indirect and direct methods to access the influence of the released toxins and surface of the material on the cell condition.

Effect of Selected Crosslinking and Stabilization Methods on the Properties of Porous Chitosan Composites Dedicated for Medical Applications

Chitosan is one of the most commonly employed natural polymers for biomedical applications. However, in order to obtain stable chitosan biomaterials with appropriate strength properties, it is necessary to subject it to crosslinking or stabilization. Composites based on chitosan and bioglass were prepared using the lyophilization method. In the experimental design, six different methods were used to obtain stable, porous chitosan/bioglass biocomposite materials. This study compared the crosslinking/stabilization of chitosan/bioglass composites with ethanol, thermal dehydration, sodium tripolyphosphate, vanillin, genipin, and sodium β-glycerophosphate. The physicochemical, mechanical, and biological properties of the obtained materials were compared. The results showed that all the selected crosslinking methods allow the production of stable, non-cytotoxic porous composites of chitosan/bioglass. The composite with genipin stood out with the best of the compared properties, taking into account biological and mechanical characteristics. The composite stabilized with ethanol is distinct in terms of its thermal properties and swelling stability, and it also promotes cell proliferation. Regarding the specific surface area, the highest value exposes the composite stabilized by the thermal dehydration method.

The Preliminary Assessment of New Biomaterials Necessitates a Comparison of Direct and Indirect Cytotoxicity Methodological Approaches

Background: Cytotoxicity testing is a primary method to establish the safety of biomaterials, e.g., biocomposites. Biomaterials involve a wide range of medical materials, which are usually solid materials and are used in bone regeneration, cardiology, or dermatology. Current advancements in science and technology provide several standard cytotoxicity testing methods that are sufficiently sensitive to detect various levels of cellular toxicity, i.e., from low to high. The aim was to compare the direct and indirect methodology described in the ISO guidelines UNE-EN ISO 10993-5:2009 Part 5. Methods: Cell proliferation was measured using WST-1 assay, and cytotoxicity was measured using LDH test kit. Results: The results indicate that the molecular surface of biomaterials have impact on the cytotoxicity and proliferation profile. Based on these results, we confirm that the indirect method does not provide a clear picture of the cell condition after the exposure to the surface, and moreover, cannot provide complete results about the effects of the material. Conclusions: Comparison of both methods shows that it is pivotal to investigate biomaterials at the very early stages using both indirect and direct methods to access the influence of the released toxins and surface of the material on the cell condition.

Porous chitosan/ZnO-doped bioglass composites as carriers of
bioactive peptides

In this study, we aimed to assess whether the composite of chitosan/ZnO-doped bioglass can be applied as a suitable scaffold for the incorporation of bioactive peptides. Material of a porous composite with 1:1 ratio of bioglass:polymer was produced and used as a matrix for delivery of peptide. A peptide with the PEPTIDES sequence (Pro-Glu-Pro-Thr-Ile-Asp-Glu-Ser) was chosen as a model peptide. Microstructure and pore sizes of chitosan/ZnO-doped bioglass were assessed. Open porosity and pore sizes of the composite were suitable for enabling the migration of cells and ensuring the easy delivery of nutrients within the implant. In addition, composite showed bioactivity and bactericidal activity against Staphylococcus aureus and Pseudomonas aeruginosa strains. Peptide alone did not have any cytotoxic activity on human fibroblasts and keratinocytes. Also it did not show any antibacterial properties and did not cause hemolysis of red blood cells. The peptide incorporated in composite showed a rapid release in the kinetics profile. The obtained results indicate that there is the technological possibility to incorporate peptides in chitosan/ ZnO-doped bioglass scaffolds. Such biomaterials have potential application in bone tissue engineering.

Proteins, peptides and peptidomimetics as active agents in implant
surface functionalization

The recent impact of implants on improving the human life quality has been enormous. During the past two decades we witnessed major advancements in both material and structural development of implants. They were driven mainly by the increasing patients’ demand and the need to address the major issues that come along with the initially underestimated complexity of the bone-implant interface.While both, the materials and design of implants reached a certain, balanced state, recent years brought a shift in focus towards the bone-implant interface as theweakest link in the increasing implant long-term usability. As a result, several approacheswere developed. They aimed at influencing and enhancing the implant osseointegration and its proper behavior when under load and stress.With this review,we would like to discuss the recent advancements in the field of implant surface modifications, emphasizing the importance of chemical methods, focusing on proteins, peptides and peptidomimetics as promising agents for titanium surface coatings.

Spectroscopic Methods Used in Implant Material Studies

It is recognized that interactions between most materials are governed by their surface properties and manifest themselves at the interface formed between them. To gain more insight into this thin layer, several methods have been deployed. Among them, spectroscopic methods have been thoroughly evaluated. Due to their exceptional sensitivity, data acquisition speed, and broad material tolerance they have been proven to be invaluable tools for surface analysis, used by scientists in many fields, for example, implant studies. Today, in modern medicine the use of implants is considered standard practice. The past two decades of constant development has established the importance of implants in dentistry, orthopedics, as well as extended their applications to other areas such as aesthetic medicine. Fundamental to the success of implants is the knowledge of the biological processes involved in interactions between an implant and its host tissue, which are directly connected to the type of implant material and its surface properties. This review aims to demonstrate the broad applications of spectroscopic methods in implant material studies, particularly discussing hard implants, surface composition studies, and surface–cell interactions.

Assessment of the Toxicity of Biocompatible Materials Supporting Bone Regeneration: Impact of the Type of Assay and Used Controls

Assessing the toxicity of new biomaterials dedicated to bone regeneration can be difficult. Many reports focus only on a single toxicity parameter, which may be insufficient for a detailed evaluation of the new material. Moreover, published data frequently do not include control cells exposed to the environment without composite or its extract. Here we present the results of two assays used in the toxicological assessment of materials’ extracts (the integrity of the cellular membrane and the mitochondrial activity/proliferation), and the influence of different types of controls used on the obtained results. Results obtained in the cellular membrane integrity assay showed a lack of toxic effects of all tested extracts, and no statistical differences between them were present. Control cells, cells incubated with chitosan extract or chitosan-bioglass extract were used as a reference in proliferation calculations to highlight the impact of controls used on the result of the experiment. The use of different baseline controls caused variability between obtained proliferation results, and influenced the outcome of statistical analysis. Our findings confirm the thesis that the type of control used in an experiment can change the final results, and it may affect the toxicological assessment of biomaterial.

Methods for crosslinking and stabilization of chitosan structures for potential medical applications

Chitosan is a well-known polymer widely used in tissue engineering and regenerative medicine. It is biocompatible, biodegradable, non-toxic, has antibacterial and osteoconductive properties. Chitosan is often used in the form of composites (with the participation of ceramic particles), membranes, hydrogels or nanoparticles. The problem with biomaterials is their low durability, rapid degradation, poor mechanical properties and cytotoxicity. Cross-linking or stabilization of such materials allows for solving these problems. It is important that the compounds used for this purpose exhibit limited or no toxicity. The presented article is a review and presents some methods of cross-linking/stabilization of chitosan structures. The analysis concerns low or non-cytotoxic cross-linking/stabilization methods. The discussed compounds used for the purpose of chitosan structure fixation are: cinnamaldehyde, genipin, L-aspartic acid, vanillin, sodium carbonate, sodium alginate, BGP, ethanol and TPP. There is discussed also a hydrothermal/dehydrothermal method which seems to be promising as it is more advantageous since no additional compounds are introduced into the structure.